Sulfate transferase and dna encoding this enzyme

ABSTRACT

A glycosaminoglycan 6-O-sulfotransferase having an activity of transferring sulfate to a hydroxyl at position 6 of a glycosamine residue of a glycosaminoglycan, which has a ratio of relative activities to substrates satisfying completely desulfated N-acetylated (CDSNAc) heparin/completely desulfated N-resulfated (CDSNS) heparin≧0.05 and a molecular weight as calculated from constituent amino acids of from 53,000 to 58,000 daltons.

TECHNICAL FIELD

[0001] The present invention relates to a sulfotransferase and to DNAencoding it. More particularly, the present invention relates to apolypeptide of 6-O-sulfotransferase which selectively sulfates thehydroxyl at position 6 of a glucosamine residue contained in aglycosaminoglycan which is a sulfate acceptor, and to DNA encoding thepolypeptide.

BACKGROUND ART

[0002] Heparin and heparan sulfate are glycosaminoglycans having arepeating unit (4GlcAβ1/IdoAα1→4GlcNAcα1→) composed of two sugars, i.e.,a hexuronic acid (HexA) residue (D-glucuronic acid (GlcA) or L-iduronicacid (IdoA) residue) and an N-acetylglucosamine (GlcNAc) residue as abasic skeleton (heparin skeleton), basically with a portion of hydroxylat position 2 of the hexuronic acid residues and a portion of aminogroups at position 2 or hydroxyl at position 6 of theN-acetylglucosamine residue being replaced with sulfates.

[0003] Cloning of the gene of an enzyme which transfers sulfate toglycosaminoglycans (glycosaminoglycan sulfotransferase) has made itpossible to readily obtain the enzyme in amounts sufficient enough toobtain information on the biosynthesis of glycosaminoglycans havingsulfates (sulfated glycosaminoglycans). This will presumably provide auseful approach to investigation on the relationship between thestructure and function of sulfated glycosaminoglycans. It has been knownthat the biosynthesis of sulfated glycosaminoglycans, in particular,biosynthesis of heparin and heparan sulfate, is achieved by a variety ofsulfation processes (Kobata, H., Hakomori, S., Nagai, K.,Glycotechnology (5), 57 (1994), published by Kodansha Scientific). Thissulfation may involve various glycosaminoglycan sulfotransferases. Asthe glycosaminoglycan sulfotransferases which transfer sulfate toheparin or heparan sulfate, heparan sulfate N-sulfotransferase(hereinafter in some cases abbreviated as “HSNST”) which catalyzesde-N-acetylation of N-acetylglucosamine residue and sulfate transfer;heparan sulfate 2-O-sulfotransferase (hereinafter in some casesabbreviated as “HS2ST”) which transfers sulfate to hydroxyl at position2 of hexuronic acid residue; and heparan sulfate 6-O-sulfotransferase(hereinafter in some cases abbreviated as “HS6ST”) which transferssulfate to hydroxyl at position 6 of N-sulfated glycosamine residue,have been isolated and for some of the sulfotransferases, cDNA cloninghas already been performed.

[0004] The inventors of the present invention have already purifiedHS6ST which selectively transfers sulfate from 3′-phosphoadenosine5′-phosphosulfate which is the sulphate donor, to hydroxyl at position 6of N-sulfated glucosamine residue contained in heparan sulfate which isthe sulfate acceptor, from cultured cells derived from Chinese hamster,mouse, and human (J. Biol. Chem., 270, 4172-4179 (1995)), have completedcloning of the enzyme (J. Biol. Chem., 273, 9208-9213 (1998)), and havesucceeded in further cloning two of the isoforms in mouse (J. Biol.Chem., 275, 28592868 (2000)).

DISCLOSURE OF THE INVENTION

[0005] Known HS6ST which selectively transfers sulfate to a hydroxyl atposition 6 of an N-sulfated glucosamine residue contained in heparansulfate exhibits a strong enzymatic activity to CDSNS (completelydesulfated N-resulfated) heparin but a very weak sulfate transferactivity to sugar chains having substantially no sulfate in amino groupsin a glucosamine residue such as CDSNAc (completely desulfatedN-acetylated) heparin, which limits modification of the heparin skeletalstructure by using the enzyme. In order to obtain many types of modifiedforms, sulfotransferases having different substrate specificities areneeded. Therefore, an object of the present invention is to providenovel glycosaminoglycan sulfotransferases which selectively sulfatehydroxyl at position 6 of a glucosamine residue of the heparin skeletonand which also exhibit sufficient activities to CDSNAc heparin.

[0006] The inventors of the present invention extensively searchedcorresponding DNAs of enzymes in humans from human cDNA based on thenucleotide sequence of DNA of one of isoforms of the above-mentionedHS6ST in mouse (hereinafter referred to as “mHS6ST2”). Surprisingly, theinventors have found that besides the corresponding human isoform havinga high homology to mHS6ST2 (hereinafter referred to as “hHS6ST2”),another enzyme having a different substrate specificity to that ofmHS6ST2 and having a homology to the hHS6ST2 is expressed and confirmedthat this enzyme has different properties to those of knownsulfotransferases, thereby achieving the present invention.

[0007] Thus, the present invention provides the followings:

[0008] (1) A glycosaminoglycan 6-O-sulfotransferase having an activityof transferring sulfate to a hydroxyl at position 6 of a glycosamineresidue of a glycosaminoglycan, which has a ratio of relative activitiesto substrates satisfying Completely Desulfated N-Acetylated (CDSNAc)heparin/Completely Desulfated N-Sulfated (CDSNS) heparin≧0.05 and amolecular weight as calculated from constituent amino acids of from53,000 to 58,000 daltons.

[0009] (2) A glycosaminoglycan 6-O-sulfotransferase having an activityof transferring sulfate to a hydroxyl at position 6 of a glycosamineresidue of a glycosaminoglycan, which has a ratio of relative activitiesto substrates satisfying N-Sulfated (NS) heparosan/Completely DesulfatedN-Sulfated (CDSNS) heparin≧1.90 and a molecular weight as calculatedfrom constituent amino acids of from 53,000 to 58,000 daltons.

[0010] (3) A glycosaminoglycan 6-O-sulfotransferase having an activityof transferring sulfate to a hydroxyl at position 6 of a glycosamineresidue of a glycosaminoglycan, which comprises a polypeptide having anamino acid sequence of SEQ ID NO: 2 or an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of SEQ ID NO: 2.

[0011] (4) An enzyme according to the item (3), in which the polypeptidehas a molecular weight of 53,000 to 58,000 daltons.

[0012] (5) An enzyme according to the item (3) or (4), in which theratio of relative activities to substrates satisfies N-Sulfated (NS)heparosan/Completely Desulfated N-Sulfated (CDSNS) heparin≧1.90.

[0013] (6) A polypeptide of the following {1} or {2}:

[0014] {1} a polypeptide having an amino acid sequence of SEQ ID NO: 2;and

[0015] {2} a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide of {1} andhaving the same antigenicity as that of the polypeptide of {1} or havingan enzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan.

[0016] (7) A polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of SEQ ID NO: 2, in which thepolypeptide has an enzymatic activity to transfer sulfate to hydroxyl atposition 6 of a glycosamine residue in a glycosaminoglycan and also hasa ratio of relative activities to substrates satisfying N-Sulfated (NS)heparosan/Completely Desulfated N-Sulfated (CDSNS) heparin≧1.90.

[0017] (8) A DNA encoding the polypeptide of the enzyme as defined inany one of the items (1) to (5) or the polypeptide as defined in theitem (6) or (7).

[0018] (9) A DNA encoding a polypeptide of the following {1} or {2}:

[0019] {1} a polypeptide having an amino acid sequence of SEQ ID NO: 2;and

[0020] {2} a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide of {1} andhaving the same antigenicity as that of the polypeptide of {1} or havingan enzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan.

[0021] (10) A DNA of any one of the followings (a) to (d):

[0022] (a) a DNA having a nucleotide sequence of SEQ ID NO: 1;

[0023] (b) a DNA having a nucleotide sequence of nucleotide residues 2to 1381 in SEQ ID NO: 1;

[0024] (c) a DNA having a nucleotide sequence which is complementary tothe nucleotide sequence of the DNA of (a) or (b); and

[0025] (d) a DNA which hybridizes with the DNA of (a), (b), or (c) understringent conditions,

[0026] the DNA encoding a polypeptide which has an enzymatic activity totransfer sulfate to hydroxyl at position 6 of a glycosamine residue in aglycosaminoglycan.

[0027] (11) A DNA according to the item (10), in which a polypeptidewhich the DNA encodes has a ratio of relative activities to substratessatisfying N-Sulfated (NS) heparosan/Completely Desulfated N-Sulfated(CDSNS) heparin≧1.90.

[0028] (12) A recombinant vector comprising the DNA as defined in anyone of the items (8) to (11).

[0029] (13) A transformant which is transformed with the recombinantvector as defined in the item (12).

[0030] (14) A method for producing the enzyme as defined in any one ofthe items (1) to (5), comprising culturing the transformant as definedin the item (13) and isolating the enzyme as defined in the item (1) or(2).

[0031] (15) A polypeptide of the following {3} or {4}:

[0032] {3} a polypeptide having an amino acid sequence of SEQ ID NO: 4;and

[0033] {4} a polypeptide having of an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide described in{3} and having an enzymatic activity to transfer sulfate to hydroxyl atposition 6 of a glycosamine residue in a glycosaminoglycan or having thesame antigenicity as that of the polypeptide described in {3}.

[0034] (16) A DNA encoding a polypeptide of the following {3} or {4}:

[0035] {3} a polypeptide having an amino acid sequence of SEQ ID NO: 4;and

[0036] {4} a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide of {3} andhaving an enzymatic activity to transfer sulfate to hydroxyl at position6 of a glycosamine residue in a glycosaminoglycan or having the sameantigenicity as that of the polypeptide described in {3} above.

[0037] (17) A DNA of any one of (e) to (h):

[0038] (e) a DNA having a nucleotide sequence of SEQ ID NO: 3;

[0039] (f) a DNA consisting of a nucleotide sequence of nucleotideresidues 15 to 1514 in SEQ ID NO: 3;

[0040] (g) a DNA having a nucleotide sequence which is complementary tothe nucleotide sequence of the DNA of (e) or (f); and

[0041] (h) a human-derived DNA which hybridizes with the DNA in (e),(f), or (g) above under stringent conditions.

[0042] (18) A method for detecting tumorigenesis of a tissue, comprisingrelating an expression amount of the polypeptide as defined in the item(6), (7), or (15) to presence of a tumor in the tissue.

[0043] (19) A method for detecting tumorigenesis of a tissue, comprisingrelating an expression amount of mRNA generated by transcription of theDNA as defined in the item (10) or (17) to presence of a tumor in thetissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 shows results of digestion of heparan sulfates modifiedwith human HS6ST isoforms, i.e., hHS6ST2, hHS6ST2v, and hHS6ST1, byheparin-degrading enzymes and fractionation by high performance liquidchromatography. A: hHS6ST1, B: hHS6ST2, C: hHS6ST2v.

[0045]FIG. 2 shows results of comparisons of the ability to formsulfated clusters among hHS6ST2, hHS6ST2v, and hHS6ST1 based on theresults shown in FIG. 1.

[0046]FIG. 3 is a photograph showing results of analysis of hHS6ST2expression by a hybridization method using a total RNA dot blottingmembrane for human tumor tissues.

[0047]FIG. 4 is a photograph showing results of analyses of expressionof hHS6ST2 and hHS6ST2v, or hHS6ST2 by a hybridization method using atotal RNA dot blotting membrane for healthy human tissues. Left: hHS6ST2and hHS6ST2v, right: hHS6ST2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0048] Hereinafter, embodiments of the present invention are described.

[0049] <1>Enzyme of the Present Invention

[0050] The enzyme of the present invention is a glycosaminoglycan6-O-sulfotransferase having an activity of transferring sulfate to ahydroxyl at position 6 of a glycosamine residue of a glycosaminoglycan,and has at least one of the followings properties (1) to (4):

[0051] (1) A ratio of relative activities to substrates satisfyingCompletely Desulfated N-Acetylated (CDSNAc) heparin/CompletelyDesulfated N-Sulfated (CDSNS) heparin≧0.05;

[0052] (2) A molecular weight as calculated from constituent amino acidsbeing from 53,000 to 58,000 daltons;

[0053] (3) A ratio of relative activities to substrates satisfyingN-Sulfated (NS) heparosan/Completely Desulfated N-Sulfated (CDSNS)heparin≧1.90; and

[0054] (4) Its polypeptide having an amino acid sequence of SEQ ID NO: 2or an amino acid sequence including substitution, deletion, insertion,or translocation of one or a few amino acids in the amino acid sequenceof SEQ ID NO: 2.

[0055] The enzyme of the present invention usually has the followingphysical and chemical properties:

[0056] (1) Action:

[0057] It selectively transfers sulfate from a sulfate donor to hydroxylat position 6 of a glucosamine residue contained in a sulfate acceptor.

[0058] (2) Substrate Specificity:

[0059] It transfers sulfate to hydroxyl at position 6 of a glucosamineresidue in CDSNS heparin (Completely Desulfated, N-Sulfated Heparin),CDSNAC heparin (Completely Desulfated, N-Acetylated Heparin), N-Sulfated(NS) heparosan, and heparin, but it does not transfer sulfate tochondroitin sulfate A and chondroitin sulfate C. A sulfate transferactivity to NDSNAc heparin is not less than 5%, preferably not less than6%, and more preferably not less than 7% based on that to CDSNS heparin.That is, a ratio of the sulfate transfer activity to CDSNAc heparin tothe sulfate transfer activity to CDSNS heparin (CDSNAc heparin/CDSNSheparin) is not less than 0.05, preferably not less than 0.06, and morepreferably not less than 0.07. Also, a sulfate transfer activity to NSheparosan is not less than 190%, and more preferably not less than 200%based on that to CDSNS heparin. That is, a ratio of the sulfate transferactivity to NS heparosan to the sulfate transfer activity to CDSNSheparin (NS heparosan/CDSNS heparin) is not less than 1.90 andpreferably not less than 2.00.

[0060] (3) Molecular Weight:

[0061] Calculated amount of the molecular weight of the polypeptidewhich constitutes the enzyme (value calculated from the amino acidsequence) is 53,000 Da or more and 58,000 Da or less.

[0062] The physical and chemical properties described above can bemeasured according to the method described in J. Biol. Chem. 270,4172-4179 (1995).

[0063] Although the enzyme of the present invention may be derived fromany mammalian so far as it has the above-mentioned properties, it isparticularly preferred that the enzyme is derived from human.

[0064] The sulfate donor for the enzyme of the present inventionpreferably includes active sulfate (3′-phosphoadenosine 5′-phosphate;hereinafter also referred to as “PAPS”).

[0065] For the enzyme of the present invention, an enzyme whosepolypeptide has an amino acid sequence of SEQ ID NO: 2 is preferable.However, if the polypeptide of an enzyme has an amino acid sequenceincluding substitution, deletion, insertion, or translocation of one ora few amino acids in the amino acid sequence of SEQ ID NO: 2, such anenzyme is also encompassed by the enzyme of the present invention so faras the enzyme having a polypeptide having such an amino acid sequencehas at least the action described in (1) above and the substratespecificity. The word “a few” in respect of amino acids in theabove-mentioned polypeptide refers to a number corresponding to 10% orless of the number of amino acids constituting the polypeptide.

[0066] One skilled in the art can easily select substitution, deletion,insertion, or translocation of one amino or more acids which will givesubstantially no damage to the enzyme activity by confirming whether ornot the targeted enzyme activity is present according to theabove-mentioned measuring method for the activity.

[0067] Preferably, the enzyme of the present invention further has thefollowing properties. That is, a sulfate transfer activity to thehydroxyl at position 6 of a glucosamine residue in heparan sulfatederived from mouse EHS tumor, when measured by the activity measuringmethod described in Examples hereinbelow, is 1.0 or more time,preferably 1.05 or more times, and particularly preferably 1.3 or moretimes that to CDSNS heparin. On the other hand, the enzyme of thepresent invention transfers substantially no sulfate to hexuronic acid.

[0068] <2> DNA1 of the Present Invention and Polypeptide Encoded by theSame

[0069] The DNA1 of the present invention is a DNA which encodes thepolypeptide of the enzyme of the present invention described in <1>above.

[0070] Specific examples of the DNA1 of the present invention include aDNA having a nucleotide sequence encoding the entire amino acid sequenceof SEQ ID NO: 2, which is the polypeptide hHS6ST2v, and DNA having anucleotide sequence encoding a polypeptide having an amino acid sequenceof SEQ ID NO: 2 including substitution, deletion, insertion, ortranslocation of one or a few amino acids and having the sameantigenicity as that of the polypeptide having the amino acid sequenceof SEQ ID NO: 2 or having an enzyme activity to transfer sulfate to thehydroxyl at position 6 of a glucosamine residue contained in aglycosaminoglycan with CDSNAc heparin/CDSNS heparin being not less than0.05, and preferably not less than 0.06. A DNA having a nucleotidesequence encoding a polypeptide whose CDSNAc heparin/CDSNS heparin isnot less than 0.07 is most preferred. However, the present invention isnot limited thereto.

[0071] Also, the DNA1 of the present invention is a DNA having anucleotide sequence encoding a polypeptide whose NS heparosan/CDSNSheparin is preferably not less than 1.90, and more preferably not lessthan 2.00.

[0072] That is, such a DNA is not limited so far as an enzyme containingthe polypeptide encoded by the DNA has the above-mentioned physical andchemical properties of the enzyme of the present invention. Specificexamples of such a DNA include a DNA having the nucleotide sequence ofSEQ ID NO: 1, a DNA having a nucleotide sequence of nucleotide residues2 to 1381 (coding region) of the nucleotide sequence of SEQ ID NO: 1,and a DNA which hybridizes with these DNAs under stringent conditions.Further examples of the DNA1 of the present invention include DNAs andRNAs having nucleotide sequences complementary to DNAs having theabove-mentioned exemplary nucleotide sequences. Furthermore, DNAs may beeither single stranded or double stranded. DNAs are not limited so faras they have the above-mentioned nucleotide sequences or nucleotidesequences complementary thereto. Also, the DNA1 of the present inventionmay contain the sequence of an intron which is to be removed beforetranslation.

[0073] As the stringent conditions described above, conditions in which50% formaldehyde, 5×SSPE (sodium chloride/sodium phosphate/EDTA(ethylenediaminetetraacetic acid) buffer), 5× Denhardt's solution, 0.5%SDS (sodium dodecylsulfate), and 100 μg/ml of denatured salmon sperm arepresent at 42° C. and conditions substantially the same as these areexemplified. That is, the stringent conditions are conditions which areused in usual hybridization of genes. Those conditions used in aNorthern blotting method, a Southern blotting method, screening usinghybridization of nucleic acids, etc. are included in the “stringentconditions” as used herein.

[0074] Substitution, deletion, insertion, or translocation of nucleotideor nucleotides in the nucleotide sequence of the DNA which causes theabove-mentioned substitution, deletion, insertion, or translocation ofone amino or more acids in an amino acid sequence can be introduced intoa DNA, for example, by synthesizing a sequence with both ends thereofbeing cleaved with a restriction enzyme and containing both sides of themutation site therein, and replacing the corresponding portion of thenucleotide sequence in a non-mutated DNA. Also, substitution, deletion,insertion, or translocation of nucleotide or nucleotides may beintroduced into DNA by a site specific mutation (Kremer, W. and Frits,H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth.In Enzymol., 154, 367 (1987)) and the like.

[0075] Note that one skilled in the art can readily understand that DNAshaving different nucleotide sequences due to degeneracy of genetic codesare also included in the DNA1 of the present invention.

[0076] Preferably, the DNA1 of the present invention has a nucleotidesequence having 95% or more homology as calculated using a gene analysiscomputer program GENETYX-MAC (manufactured by Software Development Co.,Ltd.) to the nucleotide sequence of nucleotide residues 2 to 1381 of thenucleotide sequence of SEQ ID NO: 1.

[0077] The DNA1 of the present invention is preferably DNA which isexpressed mainly in at least one of testis, ovary, and kidney as well asin brain and which is further expressed in pancreas and pituitary glandin a slight amount.

[0078] <3> Preparation Method for DNA1 of the Present Invention

[0079] The DNA1 of the present invention can be prepared byamplification by a PCR (Polymerase Chain Reaction) method or the likefrom cDNA by using primers prepared based on the nucleotide sequence ofmHS6ST2 or that of human EST or the like corresponding thereto. It canalso be prepared by artificial synthesis based on the nucleotidesequence of the DNA1 of the present invention disclosed by the presentinvention. Also, one skilled in the art can prepare the DNA1 of thepresent invention from a cDNA library or total RNA of human origin byartificially preparing primers based on the nucleotide sequence of theDNA1 of the present invention and using the primers. Furthermore, byusing the abov-mentioned primers, a DNA encoding a corresponding enzymepresent in a mammalian other than humans can be obtained from a cDNAlibrary or total RNA of the other mammalian origin.

[0080] In particular, the DNA1 of the present invention can be producedby cloning comprising the following steps:

[0081] (1) preparation of primers by using mouse HS6ST2 and human EST(Expression Sequence Tag);

[0082] (2) amplification of cDNA by a PCR method using the primersprepared in (1) above; and

[0083] (3) recovery and cloning of the PCR product.

[0084] Usually, the full-length cDNA of the present invention isselected by screening.

[0085] However, the production method for the DNA1 of the presentinvention is not limited to this and the DNA1 of the present inventioncan be produced by the above-mentioned PCR method as well as other knowncDNA cloning technique.

[0086] Hereinafter, one example of the production method for DNA of thepresent invention using cDNA of HS6ST2 of mouse origin (mHS6ST2) (J.Biol. Chem., 275, 2859-2868 (2000)) will be specifically described.

[0087] (1) Preparation of Primers by Using Mouse HS6ST2 (mHS6ST2) andHuman EST

[0088] Preparation of primers for PCR may be performed in the samemanner as in ordinary methods. However, it is preferred that anucleotide sequence of DNA of human origin be used for preparing theprimer for amplifying cDNA of human HS6ST2v. Therefore, it is preferredto search for a corresponding human EST from an EST database by usingcDNA of mHS6ST2 and prepare a primer based on the sequence of the humanEST. Examples of such an EST that can be used for the preparation ofprimers include one recorded in dbEST of GenBank under register No.AL049679. 5′ Primer and 3′ primer do not have to be prepared based onthe nucleotide sequence of human DNA. For example, one of them may beprepared based on the nuleotide sequence of cDNA of mHS6ST2 and theother may be prepared based on the nucleotide sequence of human EST.Examples of such primers include primers having nucleotide sequences ofSEQ ID NO: 5 and SEQ ID NO: 6.

[0089] (2) Amplification of cDNA by a PCR Method

[0090] By using the above-prepared primers, cDNA prepared from cells isamplified by a PCR method.

[0091] cDNA can be prepared by a conventional method. Also, acommercially available cDNA library may also be used. cDNA can beprepared, for example, by the following method.

[0092] That is, total RNA can be obtained from cultured cells or cellscollected from a tissue by, for example, a guanidine thiocyanate/CsClmethod (Kngston, R. S., (1991) in Current Protocols in MolecularBiology, Suppl. 14, Unit 4.2, Green Publishing Associates and WileyInterscience, New York) or the like. The material is not particularlylimited so far as it expresses mRNA of HS6ST2v.

[0093] From the total RNA thus obtained, poly(A)⁺RNA is purified byoligo dT (Oligo-(dT)) cellulose column chromatography or the like andfurther a reverse transcription PCR method by using a random oligonucleotide primer is performed allowing preparation of cDNA.

[0094] It is also preferred to use commercially available cDNA as theabove-mentioned cDNA in consideration of ease of operation.

[0095] Amplification of the cDNA described above by a PCR method usingthe above-mentioned primers results in specific amplification of thecDNA of HS6ST2v. The PCR method can be performed by, for example, themethod as described below.

[0096] That is, 3 μl of an aqueous solution containing a cDNA library isheated at 95° C. for 3 minutes and then immediately ice-cooled todenature cDNA. To the denatured cDNA, distilled water is added and leftto stand at 0° C. for 20 minutes or more and then the above-mentionedprimers are added. To the mixture, a reverse transcription reactionmixture, four kinds of deoxynucleotide triphosphates, and AmpliTaqpolymerase are added. The amplification reaction, which is composed of,for example, denaturation reaction at 94° C. for 30 seconds, annealingat 55° C. for 30 seconds, and elongation reaction at 72° C. for 2minutes, is repeatedly performed 35 times. Thereafter, a further 15minutes of elongation reaction is carried out. However, the conditionsof the PCR method, such as the number of repetition and temperature, maybe adjusted appropriately.

[0097] In addition, preparation of cDNA is conveniently performed with acommercially available cDNA synthesis kit. Examples of such a kitinclude Taq PCR Core Kit (manufactured by Qiagen, K.K.). Also, use of,for example, TimeSaver cDNA synthesis kit (manufactured by Pharmacia LKBBiotechnology, AB) allows coupling of cDNA to cloning vector as well assynthesis of cDNA.

[0098] Analysis of DNA amplified by such methods by separation meansaccording to molecular weight, for example, agarose gel electrophoresisreveals amplification of HS6ST2v (about 1,360 bp) and HS6ST2 (about1,500 bp).

[0099] (3) Recovery and Cloning of PCR Product

[0100] Among cDNAs amplified by the PCR method, the PCR product of about1,360 bp is recovered by a conventional method. For example, where thePCR product is fractionated by gel electrophoresis according tomolecular weight, a method of taking out DNA from gel, such as Jetsorbmay be used.

[0101] The recovered PCR product is ligated with a restriction enzymeregion and this is inserted to a cloning vector to introduce (transfect)it into a host bacterium cell by a conventional method.

[0102] The host bacterium cell to be used must be selected by a cloningvector to be used. Usually, a combination of a cloning vector whose hostis Escherichia coli (E. coli) and E. coli is frequently used. However,the present invention is not limited to this. Transfection is performedusually by mixing with E. coli of which the permeability of its cellmembrane has been changed in the presence of recombinant DNA and 30 mMcalcium chloride. In the case of λ phage vector such as λgt11,recombinant DNA may be directly introduced even into E. coli treatedwith calcium chloride. However, a method in which recombinant DNA isintroduced into the shell of the phage in a test tube in advance (thisbeing called “in vitro packaging”) to efficiently infect the recombinantDNA with E. coli is generally used. Also, packaging can be performed byusing a commercially available packaging kit (Gigapack II packagingextract: manufactured by Stratagene, etc.).

[0103] The packaged recombinant DNA is transfected into E. coli. In thisinstance, E. coli strain to be used must be selected depending on thecloning vector and plasmid vector to be used. That is, where a cloningvector containing an antibiotic resistant gene is used, E. coli shouldnot have resistance to the antibiotic. On the other hand, where acloning vector containing a gene such as P-galactosidase gene (lacZ) isused, E. coli which has no β-galactosidase activity must be selected.

[0104] This is necessary for screening for E. coli to which recombinantDNA is transfected. For example, where λgt11 cloning vector is used, itis recommended that an E. coli strain such as E. coli Y1088 be used. Onthe other hand, where Bluescript is used as a plasmid vector, E. coliJM109, etc. may be used as a host bacterium cell (indicator strain); itis just needed to suspend it together with the host bacterium cells in asoft agar medium and overlay the obtained suspension on an agar mediumso as to allow plaque formation thereon. The phage plaques holding theplasmid to which a DNA fragment has been inserted do not expressβ-galactosidase activity so that this can be easily selected.

[0105] From the above-selected clone, the introduced vector or plasmidis prepared by a conventional method and further cleaved with anappropriate restriction enzyme to obtain the DNA1 of the presentinvention. The obtained cDNA fragment, as it is or after subcloning intoan appropriate plasmid, is subjected to nucleotide sequencing.

[0106] One example of the nucleotide sequence of the DNA1 of the presentinvention thus obtained is described in SEQ ID NO: 1, and the amino acidsequence encoded thereby is described in SEQ ID NO: 2.

[0107] <4> Production Method for the Enzyme of the Present Invention

[0108] The production method for the enzyme of the present invention isa production method for the polypeptide of the enzyme or the enzyme byusing the DNA1 of the present invention.

[0109] By culturing cells transfected with the above-mentioned DNA1 ofthe present invention, allowing the enzyme of the present invention tobe generated and accumulated in the medium, and isolating thepolypeptide or the enzyme containing it from the culture, thepolypeptide or the enzyme containing it can be produced.

[0110] Cells transfected with the DNA1 of the present invention(transformants) can be obtained by inserting the DNA1 of the presentinvention into a known expression vector to construct a recombinantplasmid and performing transfection with the recombinant plasmid. Thepresent invention provides a recombinant plasmid, i.e., a recombinantvector, containing the DNA1 of the present invention and transformantsin which the DNA1 of the present invention is introduced and the DNA1 ofthe present invention is expressible and which can be used for theproduction of enzyme of the present invention and polypeptides thereof(for example, transformants transformed with the above-mentionedrecombinant vector).

[0111] Examples of cell include prokaryotic cells such as E. coli cellsand eukaryotic cells such as mammalian cells. Where prokaryotic cellssuch as E. coli cells are used, no sugar chain addition takes place tothe polypeptide of the enzyme obtained by the expression of the DNA1 ofthe present invention, so that the polypeptide of the enzyme of thepresent invention can be obtained (the obtained polypeptide isparticularly useful for producing antibodies). On the other hand, whereeukaryotic cells such as mammalian cells are used, addition of sugarchain occurs to the above-mentioned polypeptide to produce the enzyme ofthe present invention.

[0112] In the method of the present invention, host-vector systemsusually used in the production of proteins may be used. It is preferredto adopt combinations of cultured cells derived from mammalian cellssuch as COS cells (COS-1, COS-7, etc.) and 3LL-HK46 cells and expressionvectors for mammalian cells, such as pcDNAI, pME18S, pCXN2, and pCEV18.However, the present invention is not particularly limited thereto andthe enzyme of the present invention can also be produced by usingcombinations of cells of which origin is not mammalian cells withexpression vectors which can be expressed in such cells. Medium andculture conditions may be appropriately selected depending on the host,i.e., cells to be used.

[0113] By using the DNA of the present invention, it is also possible toexpress a fused polypeptide composed of the above-mentioned polypeptideand another polypeptide.

[0114] Specific examples of the construction method for a recombinantplasmid which expresses the above-mentioned fused polypeptide includethe following methods. That is, an expression plasmid vector can beconstructed by inserting the DNA of the present invention so that itcontains a protein such as protein A in the same reading region as thatof the DNA1 of the present invention and it has a plurality of proteingenes in the same reading region (for example, pGIR201protA: J. Biol.Chem. 269, 1394-1401, 1994, pcDNAI-A: J. Biol. Chem. 274, 3215-3221,1999, etc.) and a fused polypeptide can be produced by introduction ofthis expression plasmid into a host cell. In addition, a DNA fragmentwhich encodes the fused polypeptide may be cleaved out with arestriction enzyme from the expression vector and this may be ligatedwith another expression plasmid vector by the same operation asdescribed above to introduce it into host cells.

[0115] Collection of the enzyme of the present invention from theculture product can be performed by a known purification method forpolypeptides. Specifically, affinity chromatography using a Sepharosecolumn having bound thereto, for example, a substrate of theabove-mentioned enzyme, etc. may be mentioned. Where a fused polypeptideis expressed, purification can be performed by subjecting the cultureproduct of host cells to affinity chromatography or the like with anaffinity column having bound thereto a substance having high affinityfor the polypeptide (for example, antibody, etc.) fused with the enzymeof the present invention in addition to the above-mentioned affinitycolumn. Furthermore, by preliminarily incorporating, for example, alinker having an amino acid sequence which is recognized and cleaved bya specified proteolytic enzyme, between the polypeptides of the enzymeof the present invention and the other protein in the fused polypeptide,the enzyme of the present invention can be obtained by cleaving thefused polypeptide. As a combination of the proteolytic enzyme and thespecific sequence which the proteolytic enzyme recognizes includes, forexample, a combination of a signal peptidase which acts at the time ofsynthesis of proinsulin and a signal peptide of insulin. Theabove-mentioned culture product includes a medium and cultured cells.

[0116] <5> DNA2 of the Present Invention and Polypeptide Encoded Thereby

[0117] The DNA2 of the present invention is a DNA which encodes thepolypeptide of human-derived HS6ST2 (hHS6ST2). Specifically, the DNA2 ofthe present invention is a DNA which encodes a polypeptide of thefollowing (3) or (4):

[0118] (3) a polypeptide having an amino acid sequence as described inSEQ ID NO: 4, and

[0119] (4) a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion or translocation of one or a few aminoacids in the amino acid sequence of the polypeptide (3) and having anenzymatic activity of transferring sulfate from a sulfate donor tohydroxyl at position 6 of a glycosamine residue contained in a sulfateacceptor or having the same antigenicity as that of the polypeptide (3).

[0120] The DNA2 of the present invention includes, in addition to theDNA having a nucleotide sequence of SEQ ID NO: 3 or comprising anucleotide sequence of nucleotide residues 15 to 1514 thereof, a DNA ofhuman origin which hybridizes with the above-mentioned DNA understringent conditions.

[0121] The DNA2 of the present invention also includes, in addition tothe DNA having a nucleotide sequence of nucleotide residues 15 to 1514of SEQ ID NO: 3 encoding the polypeptide having the amino acid sequenceof SEQ ID NO: 4, variant DNAs by individual variation, single nucleotidepolymorphisms (SNP), etc. of the DNA of hHS6ST2.

[0122] As the DNA2 of the present invention, DNA and RNA havingnucleotide sequences complementary to the nucleotide sequence of theabove-mentioned DNA are also exemplified. Further the DNAs may be eitherdouble-stranded or single-stranded and are included in the DNA2 of thepresent invention so far as they contain the above-mentioned nucleotidesequence or those nucleotide sequences which are complementary thereto.Also, the DNA2 of the present invention may contain the sequence ofintron, which is removed before translation.

[0123] “A few” with respect to the DNA2 of the present invention is thesame as described above. One skilled in the art can measure theabove-mentioned enzymatic activity and antigenicity by a conventionalmethod by using the above-mentioned enzyme of the present invention orthe method described with respect to the DNA1 of the present inventionand select the DNA2 of the present invention which has a polypeptidehaving the enzymatic activity or the antigenicity.

[0124] Preferably, the DNA2 of the present invention has a nucleotidesequence having 95% or more homology as calculated by use of a geneanalysis computer programs GENETYX-MAC (manufactured by SoftwareDevelopment Co., Ltd.) to the nucleotide sequence of nucleotide residues15 to 1514 of the nucleotide sequence of SEQ ID NO: 3.

[0125] The DNA2 of the present invention is a DNA which is intensivelyexpressed in brain but substantially no intensive expression thereof isobserved in testis, ovary, and kidney.

[0126] <6> Preparation Method for DNA2 of the Present Invention

[0127] The DNA2 of the present invention can be prepared by recoveringan about 1,500-bp band, instead of the about 1360-bp band, from theproduct of the PCR method in the preparation method for DNA1 of thepresent invention in <4> described above.

[0128] One example of nucleotide sequence of the DNA2 of the presentinvention prepared in this manner is described in SEQ ID NO: 3 and theamino acid sequence encoded thereby is described in SEQ ID NO: 4.

[0129] The DNA2 of the present invention thus obtained may be introducedinto a recombinant vector by using the same technique as that describedin <3> above, a recombinant host cell may be prepared therewith, andhuman-derived HS6ST2 may be expressed in its culture product.

[0130] The thus prepared human-derived HS6ST2 has ability to formsulfated cluster (structure detected as ΔdiHS-tri(U,6,N):2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronicacid)-6-O-sulfo-D-glucose in disaccharide analysis) 1.2 or more timesand preferably 1.5 or more times that of human derived HS6ST2v likewiseprepared from the DNA1 of the present invention.

[0131] <7> Tumor Detection Method of the Present Invention

[0132] The tumor detection method of the present invention is adetection method for a tumor (tumorigenesis of tissue) characterized byrelating the expression amount of the polypeptide of hHS6ST2 and/orhHS6ST2v, preferably a difference in expression amount of thepolypeptide of hHS6ST2 and/or hHS6ST2v between a tumor tissue and asurrounding tissue, to the presence of a tumor in tissue cells. Inparticular, the measurement of a difference in total expression amountof hHS6ST2 and hHS6ST2v from healthy cells is preferred.

[0133] Measurement of the expression amount may be performed by directmeasurement of the amount of polypeptide or indirect measurement bymeasurement of the amount of mRNA or the like. The amount of mRNA may bemeasured by converting mRNA into cDNA by reverse transcription.

[0134] Where the amount of polypeptide is measured, the polypeptide tobe measured is usually any of polypeptides (1) to (3) described below.

[0135] (1) A polypeptide of the following {1} or {2}:

[0136] {1} a polypeptide having an amino acid sequence of SEQ ID NO: 2;and

[0137] {2} a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion or translocation of one or a few aminoacids in the amino acid sequence of the polypeptide of {1} and havingthe same antigenicity as that of the polypeptide of {1} or having anenzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan.

[0138] (2) A polypeptide having an amino acid sequence includingsubstitution, deletion, insertion or translocation of one or a few aminoacids in the amino acid sequence of SEQ ID NO: 2, in which thepolypeptide has an enzymatic activity to transfer sulfate to hydroxyl atposition 6 of a glycosamine residue in a glycosaminoglycan and also hasa ratio of relative activities to substrates satisfying N-Sulfated (NS)heparosan/Completely Desulfated N-Sulfated (CDSNS) heparin≧1.90.

[0139] (3) A polypeptide of the following {3} or {4}:

[0140] {3} a polypeptide having an amino acid sequence of SEQ ID NO: 4;and

[0141] {4} a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion or translocation of one or a few aminoacids in the amino acid sequence of the polypeptide of {3} and having anenzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan or having the sameantigenicity as that of the polypeptide of {3}.

[0142] In the case where the amount of mRNA is measured, the measuredmRNA is usually one generated by transcription of the DNA of thefollowing {1} or {2}.

[0143] (1) A DNA selected from any one of the following (a) to (d):

[0144] (a) a DNA having a nucleotide sequence of SEQ ID NO: 1;

[0145] (b) a DNA having a nucleotide sequence of nucleotide residues 2to 1381 of the nucleotide sequence of SEQ ID NO: 1;

[0146] (c) a DNA having a nucleotide sequence which is complementary tothe nucleotide sequence of the DNA of (a) or (b);

[0147] (d) a DNA which hybridizes with the DNA of (a), (b) or (c) aboveunder stringent conditions,

[0148] the DNA encoding a polypeptide which has an enzymatic activity totransfer sulfate to hydroxyl at position 6 of a glycosamine residue in aglycosaminoglycan.

[0149] (2) A DNA selected from any one of the following (e) to (h):

[0150] (e) a DNA having a nucleotide sequence of SEQ ID NO: 3;

[0151] (f) a DNA having a nucleotide sequence of nucleotide residues 15to 1514 of the nucleotide sequence of SEQ ID NO: 3;

[0152] (g) a DNA having a nucleotide sequence which is complementary tothe nucleotide sequence of the DNA of (e) or (f); and

[0153] (h) a human-derived DNA which hybridizes with the DNA of (e), (f)or (g) above under stringent conditions.

[0154] According to one embodiment of the present invention, a tissuehaving a high possibility of containing a tumor and a healthy tissuesurrounding it are collected by, for example, biopsy, etc., from whichcDNAs are prepared in accordance with a conventional method. Theexpression amounts of hHS6ST2 and hHS6ST2v are compared, for example, bya hybridization method using a DNA having a nucleotide sequence ofresidues 15 to 1514 of the nucleotide sequence of SEQ ID NO: 3 as aprobe. The results of the comparison and presence of tumor are relatedto each other and the presence of tumor is detected.

[0155] For example, in brain, small intestine, kidney and soft tissue,where a sample tissue has decreased expression amounts of polypeptidesof hHS6ST2 and hHS6ST2v as compared with those of healthy tissue, it canbe judged that a tumor is present in that tissue. On the other hand, inlung glandular tissue, large intestine, and adrenal gland, where asample tissue has increased expression amounts of polypeptides ofhHS6ST2 and hHS6ST2v as compared with those of healthy tissue, it can bejudged that a tumor is present in that tissue.

EXAMPLE

[0156] Hereinafter, the present invention will be described in moredetail by examples.

Example 1 Preparation of DNA of the Present Invention

[0157] (1) Preparation of Primer and Amplification of DNA

[0158] By using the amino acid sequence of mouse HS6ST2 (mHS6ST2) (J.Biol. Chem., 275, 2859-2868 (2000)), dbEST of GenBank was searched tofind a human counterpart DNA having a homology to encoded amino acidsequence (GenBank accession No. AL049679). A primer (Pr1: SEQ ID NO: 5)having a nucleotide sequence of nucleotide residues (−14) to (6) of thenucleotide sequence encoding mHS6ST (J. Biol. Chem., 275, 2859-2868(2000)) as counted taking the translation initiation site as 1 and aprimer (Pr2: SEQ ID NO: 6) having a nucleotide sequence complementary toa nucleotide sequence of nucleotide residues 1481-1500 of the nucleotidesequence of GenBank accession No. AL049679 were synthesized. With these,DNA was amplified by a PCR method with human brain cDNA manufactured byStratagene as a template and Taq PCR Core Kit (manufactured by Qiagen,K.K.). The PCR method was performed under conditions of denaturationreaction at 94° C. for 30 seconds, annealing at 55° C. for 30 secondsand elongation reaction at 72° C. for 2 minutes. This was performed in35 cycles. Thereafter, elongation reaction was performed for further 15minutes. Upon analysis of the amplification product obtained by thisoperation by Agarose gel electrophoresis, two amplified DNA bands ofabout 1,360 bp and of about 1,500 bp were detected.

[0159] (2) Subcloning of PCR Product and Determination of NucleotideSequence

[0160] From the gel described above, DNA present in each band wasrecovered by using Jetsorb (manufactured by Genomed, Inc.). Thenucleotide sequence of the amplified DNA was directly determined fromthe recovered DNA. Nucleotide sequences were determined by usingdGTP/deazaGTP kit containing Sequenase version 2.0 (U.S. BiochemicalCorporation). The nucleotide sequences were edited and analyzed by meansof a computer program GENETYX-MAC. It was revealed that the PCR productshave open reading frames, respectively. The determined nucleotidesequences are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, andamino acid sequences encoded by the open reading frames are shown in SEQID NO: 2 and SEQ ID NO: 4, respectively.

[0161] The larger PCR product was revealed to be a cDNA of hHS6ST2having 98% homology to mHS6ST (DNA2 of the present invention) and thesmaller PCR product had a nucleotide sequence corresponding to cDNA ofhHS6ST2 but lacking 117 base pairs present in the intermediate portionthereof, which was revealed to be a variant (hHS6ST2v) of hHS6ST2 (DNA1of the present invention). The 117 base pairs which are present inhHS6ST2 and in which hSH6ST2v is deficient were found to be a nucleotidesequence corresponding to the second and third exons of mHS6ST2 bycomparison with the results of exon/intron analysis of a genomic DNA ofmHS6ST2.

[0162] Results of comparisons of the polypeptides of hHS6ST2, hHS6ST2vand mHS6ST2 are shown in Table 1. Homology was calculated based onmHS6ST2 as a standard by using a gene analysis computer programGENETYX-MAC (manufactured by Software Development Co., Ltd.). TABLE 1Comparison of polypeptide hHS6ST2 hHS6ST2v mHS6ST2 Number of amino acids499 459 506 Molecular weight 57696 53459 58092 N-Linked glycosylation 98 9 site Homology to mHS6ST-2 92% 86% 100% PAPS-binding site PresentPresent Present

Example 2 Preparation of the Enzyme of the Present Invention

[0163] (1) Preparation of Plasmid for Expression of the Enzyme of thePresent Invention

[0164] To express the cDNA of hHS6ST2, a cDNA fragment was inserted intoan expression vector to construct a recombinant plasmid. The isolatedcDNA of hHS6ST2v was introduced into HindIII/EcoRI site of pFLAG-CMV-2(manufactured by Eastman Kodak Company), which was an expression vectorfor a mammalian, to construct a recombinant plasmid,pFLAG-CMV-2hHS6ST2v. This plasmid was constructed so as to express afused polypeptide of FLAG which is a sequence as a tag having noactivity of enzyme or the like and hHS6ST2v.

[0165] (2) Transient Expression of cDNA of hHS6ST2v in COS-7 Cells

[0166] As a host for expressing cDNA of hHS6ST2v, COS-7 cells were used.COS-7 cells were suspended in 3 ml of Dulbecco's modified Eagle medium(manufactured by Life Technologies, Inc.) containing 50 units/ml ofpenicillin, 50 μg/ml of streptomycin, and 10% (v/v) fetal calf serum andtransfected with the expression vector produced as described above byDEAE-dextran method (Aruffo, A., Current Protocols in Molecular Biology,1992, Supplement 14, Unit 16.13.1-16.13.7, Green PublishingAssociates/Wiley-Interscience, New York), and thereafter, cultured underthe condition of 37° C.

[0167] The cells transfected with pFLAG-CMV-2hHS6ST2v were cultured for67 hours and from these cells a cell extract was prepared by the methoddescribed in Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol.Chem. 270, 4172-4179. From the cell extract, hHS6ST2v was purified andisolated by affinity chromatography with an anti-FLAG antibody and itssubstrate specificity was studied (Table 2). The measurement of activitywas performed according to the method described in Habuchi, H., Habuchi,O., and Kimata, K. (1995) J. Biol. Chem. 270, 4172-4179 and Kobayashi,M., Habuchi, H., Habuchi, O., Saito, M., and Kimata, K. (1996) J. Biol.Chem. 271, 7645-7653, by using various sulfate acceptors as substratesand measuring enzymatic activity on each of them. The results are shownin relative values taking the activity on CDSNS heparin as 100. Ascontrols for comparison, mHS6ST2 and hHS6ST2 isolated from COS-7 cellstransfected with pFLAG-CMV-2 in which mHS6ST2 was introduced in the samemanner as hHS6ST2v described above (pFLAG-CMV-2 mHS6ST2) and COS-7 cellstransfected with pFLAG-CMV-2 in which hHS6ST2 was introduced in the samemanner (pFLAG-CMV-2hHS6ST2) by the same purification method as thatdescribed above were used. TABLE 2 Substrate specificity HS6ST activitySubstrate hHS6ST2v hHS6ST2 mHS6ST2 CDSNS heparin 100 100 100 NSheparosan 278 202 185 Heparin 2.1 2.4 8.6 CDSNAc heparin 7.5 8.2 4.7Heparan sulfate 91 84 65 (porcine artery) Heparan sulfate 155 106 97(Mouse EHS tumor)

[0168] It becomes evident that hHS6ST2v has higher sulfotransferaseactivities on NS heparosan, heparin, and CDSNAc heparin than those ofmHS6ST2.

[0169] Heparan sulfate from porcine artery was weighed so as to contain25 nmol of uronic acid. 50 pmol of radioactivity-labeled active sulfate({³⁵S}PAPS) and 0.35 U (amount necessary for sulfating CDSNS heparin ina rate of 0.35 pmol/min) of hHS6ST2 (prepared in the same manner as thehHS6ST2v described above with a cDNA of hHS6ST2), hHS6ST2v, or HS6ST1(J. Biol. Chem., Vol. 270 (1995), pp. 4172-4179) as a control forcomparison was added thereto and reaction was carried out at 20° C. for1 hour. After boiling the reaction mixture to inactivate the enzyme,chondroitin sulfate A (CSA: manufactured by Seikagaku Corporation) wasadded and unreacted {³⁵S}PAPS was removed by an ethanol precipitationmethod. Here, heparan sulfate modified with HS6ST2 was named HS2,heparan sulfate modified with HS6ST2v was named HS2v, and heparansulfate modified with HS6ST1 was named HS1.

[0170] HS2, HS2v, and HS1 were fractionated by a combination ofdigestion with heparin-degrading enzymes and high performance liquidchromatography. That is, 1.0 μg of the test substance was dissolved in25 μg of 20 mM sodium acetate (pH 7.0) containing 2 mM of calciumacetate. Each 1.5 mU of heparitinase and of heparitinase I as well as0.15 mU of heparitinase II (all manufactured by Seikagaku Corporation)were added thereto and reaction was carried out at 37° C. for 1 hour.

[0171] The reaction mixtures were analyzed by using HPLC (manufacturedby Waters Corporation) under the following conditions. That is, asilica-based amino column (manufactured by YMC Corporation; YMC-PackPolyamine-II column φ 4.0×250 mm) was used, and elution was performed bya concentration gradient method with sodium dihydrogen phosphate (250mM→540 mM) at a flow rate of 1.2 ml/minute according to the method ofHabuchi, et al. (Habuchi, et al. (1992) Biochem. J., 285, pp.805-813),and absorbance of the eluation was measured at 232 nm. Fractions from 16minutes to 43 minutes were recovered and then the radioactivity in eachfraction was measured by a scintillation counter (FIG. 1). In FIG. 1,ΔDiHS-6S indicates2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronicacid)-6-O-sulfo-D-glucose; ΔDiHS-NS indicates2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronicacid)-D-glucose; ΔDiHS-di(6,N)S indicates2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronicacid)-6-O-sulfo-D-glucose; ΔDiHS-di(N,U)S indicates2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronicacid)-D-glucose; and ΔDiHS-tri(U,6,N)S indicates2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronicacid)-6-O-sulfo-D-glucose. From the count number of the scintillationcounter, hHS6ST2, hHS6ST2v, and hHS6ST1 were compared by the peak ofunsaturated disaccharide (Δdi-(N,6,U)triS) having the structure in which2-O-sulfated uronic acid and 6,N-sulfated glucosamine are bound (thepeak indicating ability to form sulfated cluster), standardized by thepeak of N-acetylglucosamine residue having a sulfate group at position 6(peak at 19 minutes) (FIG. 2). As a result, it was revealed thathHS6ST2v had an ability to form sulfated cluster to an extent moderatebetween hHS2ST2 and hHS6ST1.

Example 3 Observation of HS6ST2 Expression in Tumors

[0172] By using a total RNA dot blotting membrane for human tumortissues (manufactured by Bio Chain, Inc.), expression of HS6ST2 wasanalyzed by a hybridization method (expression hybridization method)with a portion of the nucleotide sequence of the coding region ofhHS6ST2 (nucleotide residues 15-1514 of the nucleotide sequence of SEQID NO: 3) (FIG. 3). The arrangement of tissues on the blotting membraneis as shown in Table 3. Under the conditions used, total expressionamounts of hHS6ST2 and hHS6ST2v are detected. TABLE 3 Arrangement oftissues on blot membrane 1&2 3&4 5&6 7&8 9&10 11&12 A BrainPharynx/Throat Duodenum Gallbladder Testis Thyroid gland 1. Astrocytoma3. Squamous cell 5. Tumor 7. Tumor  9. Tumor 11. Follicular 2. Normalcarcinoma 6. Normal 8. Normal 10. Normal adenocarcinoma 4. Normal 12.Normal B Brain Esophagus Small Intestine Pancreas Ovary Thyroid gland 1.Neurilemmoma 3. Poorly differentiated 5. Tumor 7. Tumor  9.Adenocarcinoma 11. Follicular 2. Normal squamous cell carcinoma 6.Normal 8. Normal 10. Normal adenoma 4. Normal 12. Normal C BrainEsophagus Colon Parotid gland Ovary Thyroid gland 1. Meningioma 3.Moderately 5. Poorly 7. Tumor  9. Thecoma 11. Papillary 2. Normaldifferentiated squamous differentiated 8. Normal 10. Normaladenocarcinoma cell carcinoma adenocarcinoma 12. Normal 4. Normal 6.Normal D Lung Esophagus Colon Kidney Ovary Adrenal gland 1. Poorly 3.Adenocarcinoma 5. Well 7. Granular cell  9. Teratoma 11. Tumordifferentiated 4. Normal differentiated carcinoma 10. Normal 12. Normalsquamous cell adenocarcinoma 8. Normal carcinoma 6. Normal 2. Normal ELung Stomach Rectum Kidney Uterus Thymus 1. Moderately 3. squamous cell5. Poorly 7. Clear cell  9. Leiomyoma 11. Tumor differentiated carcinomadifferentiated carcinoma 10. Normal 12. Normal squamous cell 4. Normaladenocarcinoma 8. Normal carcinoma 6. Normal 2. Normal F Lung StomachRectum Bladder Uterus Lymphoma 1. Poorly 3. Poorly differentiated 5.Moderately 7. Transitional  9. Adenocarcimoma 11. Lymphomadifferentiated adenocarcinoma differentiated cell carcinoma 10. Normal12. Normal lymph adenocarcinoma 4. Normal adenocarcinoma grade II node2. Normal 6. Normal 8. Normal G Lung Stomach Liver Bladder BreastNon-Hodgkin's 1. Moderately 3. Moderately 5. Poorly 7. Transitional  9.invasive lymphoma differentiated differentiated differentiated cellcarcinoma ductal carcinoma 11. Lymphoma adenocarcinoma ademocarcinomahepatocellular grade III 10. Normal 12. Normal lymph 2. Normal 4. Normalcarcinoma 8. Normal node 6. Normal H Lung Stomach Liver Prostate BreastSoft tissue 1. Alveolar 3. Well differentiated 5. Moderately 7. Tumor 9. Fibroadenoma 11. Malignant carcinoma adenocarcinoma differentiated8. Normal 10. Normal fibrous 2. Normal 4. Normal hepatocellularhistocytoma carcinoma 12. Normal 6.Normal

[0173] As a result, it was revealed that in tumor sites in brain, smallintestine, kidney and soft tissue, the expression amounts of hHS6ST2 andhHS6ST2v were clearly decreased as compared with those in surroundinghealthy tissues while these were increased in tumor sites in lungglandular tissue, large intestine and adrenal gland. These resultssuggest a possibility that the presence of a tumor in each tissue whenthe expression amounts of hHS6ST2 and hHS6ST2v in tissue samples aredecreased as compared with those of healthy tissues in the case ofbrain, small intestine, kidney and soft tissue and when the expressionamounts of hHS6ST2 and hHS6ST2v are increased in tissue samples ascompared with those of healthy tissues in the case of lung glandulartissue, large intestine and adrenal gland.

[0174] In the same manner as described above, by using a total RNA dotblotting membrane for human healthy tissues (manufactured by Bio Chain,Inc.), expressions of HS6ST2 and HS6ST2v or expression of HS6ST2 wasanalyzed by a hybridization method (expression hybridization method)with a portion of the nucleotide sequence of the coding region ofhHS6ST2 (nucleotide residues 15-1514 of the nucleotide sequence of SEQID NO: 3) or of hHS6ST2 (nucleotide residues 525-644 of the nucleotidesequence of SEQ ID NO: 3), respectively, (FIG. 4). The arrangement oftissues on the blotting membrane is as shown in Table 4. TABLE 4 1 2 3 45 6 7 8 A Entire Amygdala Caudate Cerebellum Cerebral FrontalHippocampus Medulla brain nucleus cortex lobe oblongata B OccipitalPutamen Substantia Temporal Thalamus Subthalamic Spinal cord lobe nigralobe nucleus C Heart Aorta Skeletal Colon Bladder Uterus ProstateStomach muscle D Testis Ovary Pancreas Pituitary Adrenal ThyroidSalivary Mammary gland gland gland gland gland E Kidney Liver SmallSpleen Thymus Peripheral Lymph node Bone intestine leukocyte marrow FAppendix Lung Trachea Placenta G Fetal Fetal Fetal Fetal Fetal FetalFetal lung brain heart kidney liver spleen thymus H Yeast Yeast E. coliE. coil Poly- Human Col Human DNA Human DNA total RNA tRNA rRNA DNA r(A)DNA (100 μg) (500 μg) (100 μg) (100 μg) (100 μg) (100 μg) (100 μg) (100μg)

[0175] As a result, it was revealed that hHS6ST2 and hHS6ST2v wereexpressed intensively in central nerves (brain and its appendages),testis, ovary, kidney, and placenta (FIG. 4, left). Also, it wasrevealed that hHS6ST2 was intensively expressed particularly in a broadregion of brain (FIG. 4, right).

INDUSTRIAL APPLICABILITY

[0176] According to the present invention, novel isoforms of humansulfotransferase and DNAs encoding them are provided. Furthermore,tumorigenesis of tissues can be detected based on the expression amountsof the isoforms.

1 6 1 1381 DNA Homo sapiens CDS (2)..(1378) 1 c atg gat gag aaa tcc aacaag ctg ctg cta gct ttg gtg atg ctc ttc 49 Met Asp Glu Lys Ser Asn LysLeu Leu Leu Ala Leu Val Met Leu Phe 1 5 10 15 cta ttt gcc gtg atc gtcctc caa tac gtg tgc ccc ggc aca gaa tgc 97 Leu Phe Ala Val Ile Val LeuGln Tyr Val Cys Pro Gly Thr Glu Cys 20 25 30 cag ctc ctc cgc ctg cag gcgttc agc tcc ccg gtg ccg gac ccg tac 145 Gln Leu Leu Arg Leu Gln Ala PheSer Ser Pro Val Pro Asp Pro Tyr 35 40 45 cgc tcg gag gat gag agc tcc gccagg ttc gtg ccc cgc tac aat ttc 193 Arg Ser Glu Asp Glu Ser Ser Ala ArgPhe Val Pro Arg Tyr Asn Phe 50 55 60 acc cgc ggc gac ctc ctg cgc aag gtagac ttc gac atc aag ggc gat 241 Thr Arg Gly Asp Leu Leu Arg Lys Val AspPhe Asp Ile Lys Gly Asp 65 70 75 80 gac ctg atc gtg ttc ctg cac atc cagaag acc ggg ggc acc act ttc 289 Asp Leu Ile Val Phe Leu His Ile Gln LysThr Gly Gly Thr Thr Phe 85 90 95 ggc cgc cac ttg gtg cgt aac atc cag ctggag cag ccg tgc gag tgc 337 Gly Arg His Leu Val Arg Asn Ile Gln Leu GluGln Pro Cys Glu Cys 100 105 110 cgc gtg ggt cag aag aaa tgc act tgc caccgg ccg ggt aag cgg gaa 385 Arg Val Gly Gln Lys Lys Cys Thr Cys His ArgPro Gly Lys Arg Glu 115 120 125 acc tgg ctc ttc tcc agg ttc tcc acg ggctgg agc tgc ggg ttg cac 433 Thr Trp Leu Phe Ser Arg Phe Ser Thr Gly TrpSer Cys Gly Leu His 130 135 140 gcc gac tgg acc gag ctc acc agc tgt gtgccc tcc gtg gtg gac ggc 481 Ala Asp Trp Thr Glu Leu Thr Ser Cys Val ProSer Val Val Asp Gly 145 150 155 160 aag cgc gac gcc agg ctg aga ccg tccagg aac ttc cac tac atc acc 529 Lys Arg Asp Ala Arg Leu Arg Pro Ser ArgAsn Phe His Tyr Ile Thr 165 170 175 atc ctc cga gac cca gtg tcc cgg tacttg agt gag tgg agg cat gtc 577 Ile Leu Arg Asp Pro Val Ser Arg Tyr LeuSer Glu Trp Arg His Val 180 185 190 cag aga ggg gca aca tgg aaa gca tccctg cat gtc tgc gat gga agg 625 Gln Arg Gly Ala Thr Trp Lys Ala Ser LeuHis Val Cys Asp Gly Arg 195 200 205 cct cca acc tcc gaa gag ctg ccc agctgc tac act ggc gat gac tgg 673 Pro Pro Thr Ser Glu Glu Leu Pro Ser CysTyr Thr Gly Asp Asp Trp 210 215 220 tct ggc tgc ccc ctc aaa gag ttt atggac tgt ccc tac aat cta gcc 721 Ser Gly Cys Pro Leu Lys Glu Phe Met AspCys Pro Tyr Asn Leu Ala 225 230 235 240 aac aac cgc cag gtg cgc atg ctctcc gac ctg acc ctg gta ggc tgc 769 Asn Asn Arg Gln Val Arg Met Leu SerAsp Leu Thr Leu Val Gly Cys 245 250 255 tac aac ctc tct gtc atg cct gaaaag caa aga aac aag gtc ctt ctg 817 Tyr Asn Leu Ser Val Met Pro Glu LysGln Arg Asn Lys Val Leu Leu 260 265 270 gaa agt gcc aag tca aat ctg aagcac atg gcg ttc ttc ggc ctc act 865 Glu Ser Ala Lys Ser Asn Leu Lys HisMet Ala Phe Phe Gly Leu Thr 275 280 285 gag ttt cag cgg aag acc caa tatctg ttt gag aaa acc ttc aac atg 913 Glu Phe Gln Arg Lys Thr Gln Tyr LeuPhe Glu Lys Thr Phe Asn Met 290 295 300 aac ttt att tcg cca ttt acc cagtat aat acc act agg gcc tct agt 961 Asn Phe Ile Ser Pro Phe Thr Gln TyrAsn Thr Thr Arg Ala Ser Ser 305 310 315 320 gta gag atc aat gag gaa attcaa aag cgt att gag gga ctg aat ttt 1009 Val Glu Ile Asn Glu Glu Ile GlnLys Arg Ile Glu Gly Leu Asn Phe 325 330 335 ctg gat atg gag ttg tac agctat gcc aaa gac ctt ttt ttg cag agg 1057 Leu Asp Met Glu Leu Tyr Ser TyrAla Lys Asp Leu Phe Leu Gln Arg 340 345 350 tat cag ttt atg agg cag aaagag cat cag gag gcc agg cga aag cgt 1105 Tyr Gln Phe Met Arg Gln Lys GluHis Gln Glu Ala Arg Arg Lys Arg 355 360 365 cag gaa caa cgc aaa ttt ctgaag gga agg ctc ctt cag acc cat ttc 1153 Gln Glu Gln Arg Lys Phe Leu LysGly Arg Leu Leu Gln Thr His Phe 370 375 380 cag agc cag ggt cag ggc cagagc cag aat ccg aat cag aat cag agt 1201 Gln Ser Gln Gly Gln Gly Gln SerGln Asn Pro Asn Gln Asn Gln Ser 385 390 395 400 cag aac cca aat ccg aatgcc aat cag aac ctg act cag aat ctg atg 1249 Gln Asn Pro Asn Pro Asn AlaAsn Gln Asn Leu Thr Gln Asn Leu Met 405 410 415 cag aat ctg act cag agtttg agc cag aag gag aac cgg gaa agc ccg 1297 Gln Asn Leu Thr Gln Ser LeuSer Gln Lys Glu Asn Arg Glu Ser Pro 420 425 430 aag cag aac tca ggc aaggag cag aat gat aac acc agc aat ggc acc 1345 Lys Gln Asn Ser Gly Lys GluGln Asn Asp Asn Thr Ser Asn Gly Thr 435 440 445 aac gac tac ata ggc agtgta gag aaa tgg cgt taa 1381 Asn Asp Tyr Ile Gly Ser Val Glu Lys Trp Arg450 455 2 459 PRT Homo sapiens 2 Met Asp Glu Lys Ser Asn Lys Leu Leu LeuAla Leu Val Met Leu Phe 1 5 10 15 Leu Phe Ala Val Ile Val Leu Gln TyrVal Cys Pro Gly Thr Glu Cys 20 25 30 Gln Leu Leu Arg Leu Gln Ala Phe SerSer Pro Val Pro Asp Pro Tyr 35 40 45 Arg Ser Glu Asp Glu Ser Ser Ala ArgPhe Val Pro Arg Tyr Asn Phe 50 55 60 Thr Arg Gly Asp Leu Leu Arg Lys ValAsp Phe Asp Ile Lys Gly Asp 65 70 75 80 Asp Leu Ile Val Phe Leu His IleGln Lys Thr Gly Gly Thr Thr Phe 85 90 95 Gly Arg His Leu Val Arg Asn IleGln Leu Glu Gln Pro Cys Glu Cys 100 105 110 Arg Val Gly Gln Lys Lys CysThr Cys His Arg Pro Gly Lys Arg Glu 115 120 125 Thr Trp Leu Phe Ser ArgPhe Ser Thr Gly Trp Ser Cys Gly Leu His 130 135 140 Ala Asp Trp Thr GluLeu Thr Ser Cys Val Pro Ser Val Val Asp Gly 145 150 155 160 Lys Arg AspAla Arg Leu Arg Pro Ser Arg Asn Phe His Tyr Ile Thr 165 170 175 Ile LeuArg Asp Pro Val Ser Arg Tyr Leu Ser Glu Trp Arg His Val 180 185 190 GlnArg Gly Ala Thr Trp Lys Ala Ser Leu His Val Cys Asp Gly Arg 195 200 205Pro Pro Thr Ser Glu Glu Leu Pro Ser Cys Tyr Thr Gly Asp Asp Trp 210 215220 Ser Gly Cys Pro Leu Lys Glu Phe Met Asp Cys Pro Tyr Asn Leu Ala 225230 235 240 Asn Asn Arg Gln Val Arg Met Leu Ser Asp Leu Thr Leu Val GlyCys 245 250 255 Tyr Asn Leu Ser Val Met Pro Glu Lys Gln Arg Asn Lys ValLeu Leu 260 265 270 Glu Ser Ala Lys Ser Asn Leu Lys His Met Ala Phe PheGly Leu Thr 275 280 285 Glu Phe Gln Arg Lys Thr Gln Tyr Leu Phe Glu LysThr Phe Asn Met 290 295 300 Asn Phe Ile Ser Pro Phe Thr Gln Tyr Asn ThrThr Arg Ala Ser Ser 305 310 315 320 Val Glu Ile Asn Glu Glu Ile Gln LysArg Ile Glu Gly Leu Asn Phe 325 330 335 Leu Asp Met Glu Leu Tyr Ser TyrAla Lys Asp Leu Phe Leu Gln Arg 340 345 350 Tyr Gln Phe Met Arg Gln LysGlu His Gln Glu Ala Arg Arg Lys Arg 355 360 365 Gln Glu Gln Arg Lys PheLeu Lys Gly Arg Leu Leu Gln Thr His Phe 370 375 380 Gln Ser Gln Gly GlnGly Gln Ser Gln Asn Pro Asn Gln Asn Gln Ser 385 390 395 400 Gln Asn ProAsn Pro Asn Ala Asn Gln Asn Leu Thr Gln Asn Leu Met 405 410 415 Gln AsnLeu Thr Gln Ser Leu Ser Gln Lys Glu Asn Arg Glu Ser Pro 420 425 430 LysGln Asn Ser Gly Lys Glu Gln Asn Asp Asn Thr Ser Asn Gly Thr 435 440 445Asn Asp Tyr Ile Gly Ser Val Glu Lys Trp Arg 450 455 3 1514 DNA Homosapiens CDS (15)..(1511) 3 ccagcgtcgg gaac atg gat gag aaa tcc aac aagctg ctg cta gct ttg 50 Met Asp Glu Lys Ser Asn Lys Leu Leu Leu Ala Leu 15 10 gtg atg ctc ttc cta ttt gcc gtg atc gtc ctc caa tac gtg tgc ccc 98Val Met Leu Phe Leu Phe Ala Val Ile Val Leu Gln Tyr Val Cys Pro 15 20 25ggc aca gaa tgc cag ctc ctc cgc ctg cag gcg ttc agc tcc ccg gtg 146 GlyThr Glu Cys Gln Leu Leu Arg Leu Gln Ala Phe Ser Ser Pro Val 30 35 40 ccggac ccg tac cgc tcg gag gat gag agc tcc gcc agg ttc gtg ccc 194 Pro AspPro Tyr Arg Ser Glu Asp Glu Ser Ser Ala Arg Phe Val Pro 45 50 55 60 cgctac aat ttc acc cgc ggc gac ctc ctg cgc aag gta gac ttc gac 242 Arg TyrAsn Phe Thr Arg Gly Asp Leu Leu Arg Lys Val Asp Phe Asp 65 70 75 atc aagggc gat gac ctg atc gtg ttc ctg cac atc cag aag acc ggg 290 Ile Lys GlyAsp Asp Leu Ile Val Phe Leu His Ile Gln Lys Thr Gly 80 85 90 ggc acc actttc ggc cgc cac ttg gtg cgt aac atc cag ctg gag cag 338 Gly Thr Thr PheGly Arg His Leu Val Arg Asn Ile Gln Leu Glu Gln 95 100 105 ccg tgc gagtgc cgc gtg ggt cag aag aaa tgc act tgc cac cgg ccg 386 Pro Cys Glu CysArg Val Gly Gln Lys Lys Cys Thr Cys His Arg Pro 110 115 120 ggt aag cgggaa acc tgg ctc ttc tcc agg ttc tcc acg ggc tgg agc 434 Gly Lys Arg GluThr Trp Leu Phe Ser Arg Phe Ser Thr Gly Trp Ser 125 130 135 140 tgc gggttg cac gcc gac tgg acc gag ctc acc agc tgt gtg ccc tcc 482 Cys Gly LeuHis Ala Asp Trp Thr Glu Leu Thr Ser Cys Val Pro Ser 145 150 155 gtg gtggac ggc aag cgc gac gcc agg ctg aga ccg tcc agg tgg agg 530 Val Val AspGly Lys Arg Asp Ala Arg Leu Arg Pro Ser Arg Trp Arg 160 165 170 att tttcag att cta gat gca gca agt aag gat aaa cgg ggt tct cca 578 Ile Phe GlnIle Leu Asp Ala Ala Ser Lys Asp Lys Arg Gly Ser Pro 175 180 185 aac actaac gca ggc gcc aac tct ccg tca tcc aca aag acc cgg aac 626 Asn Thr AsnAla Gly Ala Asn Ser Pro Ser Ser Thr Lys Thr Arg Asn 190 195 200 aca tctaag agt ggg aag aac ttc cac tac atc acc atc ctc cga gac 674 Thr Ser LysSer Gly Lys Asn Phe His Tyr Ile Thr Ile Leu Arg Asp 205 210 215 220 ccagtg tcc cgg tac ttg agt gag tgg agg cat gtc cag aga ggg gca 722 Pro ValSer Arg Tyr Leu Ser Glu Trp Arg His Val Gln Arg Gly Ala 225 230 235 acatgg aaa gca tcc ctg cat gtc tgc gat gga agg cct cca acc tcc 770 Thr TrpLys Ala Ser Leu His Val Cys Asp Gly Arg Pro Pro Thr Ser 240 245 250 gaagag ctg ccc agc tgc tac act ggc gat gac tgg tct ggc tgc ccc 818 Glu GluLeu Pro Ser Cys Tyr Thr Gly Asp Asp Trp Ser Gly Cys Pro 255 260 265 ctcaaa gag ttt atg gac tgt ccc tac aat cta gcc aac aac cgc cag 866 Leu LysGlu Phe Met Asp Cys Pro Tyr Asn Leu Ala Asn Asn Arg Gln 270 275 280 gtgcgc atg ctc tcc gac ctg acc ctg gta ggc tgc tac aac ctc tct 914 Val ArgMet Leu Ser Asp Leu Thr Leu Val Gly Cys Tyr Asn Leu Ser 285 290 295 300gtc atg cct gaa aag caa aga aac aag gtc ctt ctg gaa agt gcc aag 962 ValMet Pro Glu Lys Gln Arg Asn Lys Val Leu Leu Glu Ser Ala Lys 305 310 315tca aat ctg aag cac atg gcg ttc ttc ggc ctc act gag ttt cag cgg 1010 SerAsn Leu Lys His Met Ala Phe Phe Gly Leu Thr Glu Phe Gln Arg 320 325 330aag acc caa tat ctg ttt gag aaa acc ttc aac atg aac ttt att tcg 1058 LysThr Gln Tyr Leu Phe Glu Lys Thr Phe Asn Met Asn Phe Ile Ser 335 340 345cca ttt acc cag tat aat acc act agg gcc tct agt gta gag atc aat 1106 ProPhe Thr Gln Tyr Asn Thr Thr Arg Ala Ser Ser Val Glu Ile Asn 350 355 360gag gaa att caa aag cgt att gag gga ctg aat ttt ctg gat atg gag 1154 GluGlu Ile Gln Lys Arg Ile Glu Gly Leu Asn Phe Leu Asp Met Glu 365 370 375380 ttg tac agc tat gcc aaa gac ctt ttt ttg cag agg tat cag ttt atg 1202Leu Tyr Ser Tyr Ala Lys Asp Leu Phe Leu Gln Arg Tyr Gln Phe Met 385 390395 agg cag aaa gag cat cag gag gcc agg cga aag cgt cag gaa caa cgc 1250Arg Gln Lys Glu His Gln Glu Ala Arg Arg Lys Arg Gln Glu Gln Arg 400 405410 aaa ttt ctg aag gga agg ctc ctt cag acc cat ttc cag agc cag ggt 1298Lys Phe Leu Lys Gly Arg Leu Leu Gln Thr His Phe Gln Ser Gln Gly 415 420425 cag ggc cag agc cag aat ccg aat cag aat cag agt cag aac cca aat 1346Gln Gly Gln Ser Gln Asn Pro Asn Gln Asn Gln Ser Gln Asn Pro Asn 430 435440 ccg aat gcc aat cag aac ctg act cag aat ctg atg cag aat ctg act 1394Pro Asn Ala Asn Gln Asn Leu Thr Gln Asn Leu Met Gln Asn Leu Thr 445 450455 460 cag agt ttg agc cag aag gag aac cgg gaa agc ccg aag cag aac tca1442 Gln Ser Leu Ser Gln Lys Glu Asn Arg Glu Ser Pro Lys Gln Asn Ser 465470 475 ggc aag gag cag aat gat aac acc agc aat ggc acc aac gac tac ata1490 Gly Lys Glu Gln Asn Asp Asn Thr Ser Asn Gly Thr Asn Asp Tyr Ile 480485 490 ggc agt gta gag aaa tgg cgt taa 1514 Gly Ser Val Glu Lys Trp Arg495 4 499 PRT Homo sapiens 4 Met Asp Glu Lys Ser Asn Lys Leu Leu Leu AlaLeu Val Met Leu Phe 1 5 10 15 Leu Phe Ala Val Ile Val Leu Gln Tyr ValCys Pro Gly Thr Glu Cys 20 25 30 Gln Leu Leu Arg Leu Gln Ala Phe Ser SerPro Val Pro Asp Pro Tyr 35 40 45 Arg Ser Glu Asp Glu Ser Ser Ala Arg PheVal Pro Arg Tyr Asn Phe 50 55 60 Thr Arg Gly Asp Leu Leu Arg Lys Val AspPhe Asp Ile Lys Gly Asp 65 70 75 80 Asp Leu Ile Val Phe Leu His Ile GlnLys Thr Gly Gly Thr Thr Phe 85 90 95 Gly Arg His Leu Val Arg Asn Ile GlnLeu Glu Gln Pro Cys Glu Cys 100 105 110 Arg Val Gly Gln Lys Lys Cys ThrCys His Arg Pro Gly Lys Arg Glu 115 120 125 Thr Trp Leu Phe Ser Arg PheSer Thr Gly Trp Ser Cys Gly Leu His 130 135 140 Ala Asp Trp Thr Glu LeuThr Ser Cys Val Pro Ser Val Val Asp Gly 145 150 155 160 Lys Arg Asp AlaArg Leu Arg Pro Ser Arg Trp Arg Ile Phe Gln Ile 165 170 175 Leu Asp AlaAla Ser Lys Asp Lys Arg Gly Ser Pro Asn Thr Asn Ala 180 185 190 Gly AlaAsn Ser Pro Ser Ser Thr Lys Thr Arg Asn Thr Ser Lys Ser 195 200 205 GlyLys Asn Phe His Tyr Ile Thr Ile Leu Arg Asp Pro Val Ser Arg 210 215 220Tyr Leu Ser Glu Trp Arg His Val Gln Arg Gly Ala Thr Trp Lys Ala 225 230235 240 Ser Leu His Val Cys Asp Gly Arg Pro Pro Thr Ser Glu Glu Leu Pro245 250 255 Ser Cys Tyr Thr Gly Asp Asp Trp Ser Gly Cys Pro Leu Lys GluPhe 260 265 270 Met Asp Cys Pro Tyr Asn Leu Ala Asn Asn Arg Gln Val ArgMet Leu 275 280 285 Ser Asp Leu Thr Leu Val Gly Cys Tyr Asn Leu Ser ValMet Pro Glu 290 295 300 Lys Gln Arg Asn Lys Val Leu Leu Glu Ser Ala LysSer Asn Leu Lys 305 310 315 320 His Met Ala Phe Phe Gly Leu Thr Glu PheGln Arg Lys Thr Gln Tyr 325 330 335 Leu Phe Glu Lys Thr Phe Asn Met AsnPhe Ile Ser Pro Phe Thr Gln 340 345 350 Tyr Asn Thr Thr Arg Ala Ser SerVal Glu Ile Asn Glu Glu Ile Gln 355 360 365 Lys Arg Ile Glu Gly Leu AsnPhe Leu Asp Met Glu Leu Tyr Ser Tyr 370 375 380 Ala Lys Asp Leu Phe LeuGln Arg Tyr Gln Phe Met Arg Gln Lys Glu 385 390 395 400 His Gln Glu AlaArg Arg Lys Arg Gln Glu Gln Arg Lys Phe Leu Lys 405 410 415 Gly Arg LeuLeu Gln Thr His Phe Gln Ser Gln Gly Gln Gly Gln Ser 420 425 430 Gln AsnPro Asn Gln Asn Gln Ser Gln Asn Pro Asn Pro Asn Ala Asn 435 440 445 GlnAsn Leu Thr Gln Asn Leu Met Gln Asn Leu Thr Gln Ser Leu Ser 450 455 460Gln Lys Glu Asn Arg Glu Ser Pro Lys Gln Asn Ser Gly Lys Glu Gln 465 470475 480 Asn Asp Asn Thr Ser Asn Gly Thr Asn Asp Tyr Ile Gly Ser Val Glu485 490 495 Lys Trp Arg 5 20 DNA Artificial Sequence primer 5 ccagcgtcgggaacatggat 20 6 20 DNA Artificial Sequence primer 6 gccatttaacgccatttctc 20

What is claimed is:
 1. A glycosaminoglycan 6-O-sulfotransferase havingan activity of transferring sulfate to a hydroxyl at position 6 of aglycosamine residue of a glycosaminoglycan, which has a ratio ofrelative activities to substrates satisfying Completely DesulfatedN-Acetylated heparin/Completely Desulfated N-Sulfated heparin≧0.05 and amolecular weight as calculated from constituent amino acids of from53,000 to 58,000 daltons.
 2. A glycosaminoglycan 6-O-sulfotransferasehaving an activity of transferring sulfate to a hydroxyl at position 6of a glycosamine residue of a glycosaminoglycan, which has a ratio ofrelative activities to substrates satisfying N-Sulfatedheparosan/Completely Desulfated N-Sulfated heparin≧1.90 and a molecularweight as calculated from constituent amino acids of from 53,000 to58,000 daltons.
 3. A glycosaminoglycan 6-O-sulfotransferase having anactivity of transferring sulfate to a hydroxyl at position 6 of aglycosamine residue of a glycosaminoglycan, which comprises apolypeptide having an amino acid sequence of SEQ ID NO: 2 or an aminoacid sequence including substitution, deletion, insertion ortranslocation of one or a few amino acids in the amino acid sequence ofSEQ ID NO:
 2. 4. An enzyme according to claim 3, wherein the polypeptidehas a molecular weight of 53,000 to 58,000 daltons.
 5. An enzymeaccording to claim 3 or 4, wherein the ratio of relative activities tosubstrates satisfies N-Sulfated heparosan/Completely DesulfatedN-Sulfated heparin≧1.90.
 6. A polypeptide of the following (1) or (2):(1) a polypeptide having an amino acid sequence of SEQ ID NO: 2; and (2)a polypeptide having an amino acid sequence including substitution,deletion, insertion, or translocation of one or a few amino acids in theamino acid sequence of the polypeptide of (1) and having the sameantigenicity as that of the polypeptide of (1) or having an enzymaticactivity to transfer sulfate to hydroxyl at position 6 of a glycosamineresidue in a glycosaminoglycan.
 7. A polypeptide comprising an aminoacid sequence including substitution, deletion, insertion ortranslocation of one or a few amino acids in the amino acid sequence SEQID NO: 2, wherein the polypeptide has the enzymatic activity to transfersulfate to hydroxyl at position 6 of a glycosamine residue in aglycosaminoglycan and also has a ratio of relative activities tosubstrates satisfying N-Sulfated heparosan/Completely DesulfatedN-Sulfated heparin≧1.90.
 8. A DNA encoding the polypeptide of the enzymeas defined in any one of claims 1 to 5 or the polypeptide as defined inclaim 6 or
 7. 9. A DNA encoding a polypeptide of the following (1) or(2): (1) a polypeptide having an amino acid sequence of SEQ ID NO: 2;and (2) a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide of (1) andhaving the same antigenicity as that of the polypeptide of (1) or havingan enzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan.
 10. A DNA of any one of thefollowing (a) to (d): (a) a DNA having a nucleotide sequence of SEQ IDNO: 1; (b) a DNA having a nucleotide sequence of nucleotide residues 2to 1381 in SEQ ID NO: 1; (c) a DNA having a nucleotide sequence which iscomplementary to the nucleotide sequence of the DNA of (a) or (b); and(d) a DNA which hybridizes with the DNA of (a), (b), or (c) understringent conditions, the DNA encoding a polypeptide which has anenzymatic activity to transfer sulfate to hydroxyl at position 6 of aglycosamine residue in a glycosaminoglycan.
 11. A DNA according to claim10, wherein a polypeptide which the DNA encodes has a ratio of relativeactivities to substrates satisfying N-Sulfated heparosan/CompletelyDesulfated N-Sulfated heparin≧1.90.
 12. A recombinant vector comprisingthe DNA as defined in any one of claims 8 to
 11. 13. A transformantwhich is transformed with the recombinant vector as defined in claim 12.14. A method for producing the enzyme as defined in any one of claims 1to 5, comprising culturing the transformant as defined in claim 13 andisolating the enzyme as defined in claim 1 or
 2. 15. A polypeptide ofthe following (3) or (4): (3) a polypeptide having an amino acidsequence of SEQ ID NO: 4; and (4) a polypeptide having an amino acidsequence including substitution, deletion, insertion, or translocationof one or a few amino acids in the amino acid sequence of thepolypeptide of (3) and having an enzymatic activity to transfer sulfateto hydroxyl at position 6 of a glycosamine residue in aglycosaminoglycan or having the same antigenicity as that of thepolypeptide of (3).
 16. A DNA encoding a polypeptide of the following(3) or (4): (3) a polypeptide having an amino acid sequence of SEQ IDNO: 4; and (4) a polypeptide having an amino acid sequence includingsubstitution, deletion, insertion, or translocation of one or a fewamino acids in the amino acid sequence of the polypeptide of (3) andhaving an enzymatic activity to transfer sulfate to hydroxyl at position6 of a glycosamine residue in a glycosaminoglycan or having the sameantigenicity as that of the polypeptide of (3).
 17. A DNA of any one ofthe following (e) to (h): (e) a DNA having a nucleotide sequence of SEQID NO: 3; (f) a DNA having a nucleotide sequence of nucleotide residues15 to 1514 in SEQ ID NO: 3; (g) a DNA having a nucleotide sequence whichis complementary to the nucleotide sequence of the DNA of (e) or (f);and (h) a human-derived DNA which hybridizes with the DNA of (e), (f),or (g) under stringent conditions.
 18. A method for detectingtumorigenesis of a tissue, comprising relating an expression amount ofthe polypeptide as defined in claim 6, 7 or 15 to presence of a tumor inthe tissue.
 19. A method for detecting tumorigenesis of a tissue,comprising relating an expression amount of mRNA generated bytranscription of the DNA as defined in claim 10 or 17 to presence of atumor in the tissue.